B-CELL LINES IMMORTALIZED WITH AN EPSTEIN-BARR-VIRUS MUTANT LACKING THE CP EBNA2 ENHANCER ARE BIASED TOWARD UTILIZATION OF THE ORIP-PROXIMAL EBNA GENE PROMOTER WP1

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in v itro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to b e mutually exclusive in established lymphoblastoid cell lines, Initial ly after infection, the EBNA genes are transcribed from Wp, which is p resent in multiples copies within the major internal repeat of EBV, Ap proximately 48 to 72 h postinfection, Wp is downregulated, with a corr esponding increase in transcription from Cp, An EBNA2-responsive enhan cer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously bee n proposed (Woisetschlaeger et al., Proc, Natl, Acad, Sci, USA 87:1725 -1729, 1991), To critically assess the potential role for this enhance r region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated, Lymphoblastoid cell lines were s creened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion, A quantitative S1 nuclease prot ection assay was developed to allow comparison of relative Cp and Wp a ctivities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion, In general, the wild-type recombinants had higher levels of Cp-initiated transcri pts than Wp-initiated transcripts, In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity, Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these muta nts, S1 nuclease protection assays using a probe which hybridizes to t he W2 exon, contained in both Cp-and Wp-initiated transcripts, indicat ed that the total level of transcription from Cp and Wp remained the s ame in wild-type and EBNA2 enhancer mutant cell lines, The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mu tually exclusive.